Strains and culture conditions
The following strains were used: B. cepacia LMG 1222 and LMG 18821; Burkholderia multivorans LMG 18822, LMG 18825, LMG 13010 and LMG 17588; Burkholderia cenocepacia LMG 16656, LMG 18828, LMG 18829 and LMG 18830; Burkholderia vietnamiensis LMG 10929 and LMG 18835; Burkholderia ambifaria LMG 19182 and LMG 19467; Burkholderia lata LMG 6992 and R-9940; Burkholderia stabilis LMG 14294 and LMG 14086; Burkholderia dolosa LMG 18943 and LMG 18941; Burkholderia anthina LMG 20980 and LMG 20983; Burkholderia pyrrocinia LMG 21824; Burkholderia ubonensis LMG 20358 and LMG 24263; Burkholderia latens LMG 24064; Burkholderia arboris LMG 24066 and R-132; Burkholderia seminalis LMG 24067 and LMG 24272; Burkholderia metallica LMG 24068 and R-2712; and Burkholderia contaminans LMG 16227 and R-12710. The biological and geographic origin of every Bcc strain is presented in Additional file 1. All strains were obtained from the BCCM/LMG Bacteria Collection (Ghent, Belgium) or were kindly provided by Prof. P. Vandamme (Ghent University, Belgium). Two control strains were included; P. aeruginosa ATCC 27853 and E. coli ATCC 25922, both obtained from the ATCC collection (Manassas, VA, USA). Bacterial cultures were stored at -80 °C in Microbank vials (Prolab Diagnostics, Richmond Hill, ON, Canada) and were subcultured twice on Luria-Bertani agar (LBA; LabM Limited, Heywood, UK) before use. All cultures were incubated aerobically at 37 °C.
Antibiotics and β-lactamase inhibitors
We used several β-lactam antibiotics of different classes including amoxicillin (AMOX; aminopenicillin), cefoxitin (CFX; 2nd generation cephalosporin), ceftazidime (CAZ; 3rd generation cephalosporin), cefepime (CFP; 4th generation cephalosporin), meropenem (MEM; carbapenem) and aztreonam (AZT; monobactam). The effect of combining these antibiotics with the β-lactamase inhibitors clavulanic acid (CLA), sulbactam (SUL), tazobactam (TAZ) and avibactam (AVI) was investigated. AMOX, CFX, CAZ, CFP, CLA, SUL and TAZ were purchased from Sigma-Aldrich (St. Louis, MO, USA). MEM was obtained from Hospira Benelux (Antwerp, Belgium), AZT from TCI Europe (Zwijndrecht, Belgium) and AVI was obtained from Adooq Bioscience (Irwin, CA, USA). The concentration range tested for CAZ and MEM was 0.25 − 128 mg/L. Higher concentrations were tested for AMOX, CFX, CFP and AZT; between 1 and 512 mg/L (according to Peeters et al. [4] and CLSI guidelines). The β-lactamase inhibitors were added at fixed concentrations as mentioned in the European Committee on Antimicrobial Susceptibility testing (EUCAST) breakpoint tables; 2 mg/L for CLA and 4 mg/L for SUL, TAZ and AVI [22].
MIC determination
Susceptibility of the selected Bcc strains was investigated by determining minimum inhibitory concentrations (MICs) (in triplicate) of β-lactam antibiotics in the presence or absence of β-lactamase inhibitors, according to the EUCAST broth dilution guidelines using flat-bottomed 96-well microtitre plates (TPP, Trasadingen, Switzerland) [23]. Antibiotic solutions were added to the wells and two-fold dilutions were made. Planktonic cultures were grown overnight in Luria-Bertani broth (LBB; LabM, Lancashire, UK) at 37 °C. The cultures were then adjusted with double-concentrated Mueller-Hinton broth (MHB; Beckton, Dickinson & Company (BD), Erembodegem, Belgium) to obtain a final inoculum of 5 x 105 cfu/ml. Plates were incubated for 24 h at 37 °C and optical density was determined at 590 nm using an Envision multilable plate reader (Perkin Elmer, Waltham, MA, USA). The MIC value is the lowest concentration of the antibiotic that completely inhibits bacterial growth [4, 23].
β-lactamase activity assay
We explored differences in β-lactamase activity by using a β-lactamase activity assay kit (Sigma-Aldrich, St. Louis, MO, USA). This assay is based on the hydrolysis of the chromogenic molecule nitrocefin, a non-antimicrobial cephalosporin, by β-lactamase which leads to the production of a colorimetric product. Formation of this product is monitored by measuring absorbance at 490 nm in an Envision multilable plate reader; every minute, for 60 to 90 min at 25 °C. The amount of enzyme required to hydrolyze 1.0 μmol of nitrocefin per minute at pH 7.0 at 25 °C is equal to one unit of β-lactamase [10, 11, 24]. Bacterial cultures were grown in 96-well microtitre plates in the presence or absence of antibiotics. Antibiotics were added to the microtitre plates at concentrations of ¼ MIC and the β-lactamase inhibitors SUL, TAZ and AVI were added at 4 mg/L. Planktonic cultures were grown overnight in LB broth at 37 °C, then adjusted with double-concentrated MHB to obtain a final inoculum of 5 x 105 cfu/ml in the 96-well plates. For every condition 10 wells in the same plate were filled and the plates were incubated at 37 °C for 24 h. After incubation, the content of the 10 wells was collected in a pre-weighed plastic tube and centrifuged at 10 000 RCF for 10 min. Then, supernatant was discarded and the pellet was weighed and resuspended with 5 μL of β-lactamase assay buffer per mg sample. Subsequently, samples were sonicated for 5 min, placed on ice for 5 min and centrifuged at 16 000 RCF at 4 °C for 20 min. 1 – 50 μL of the unknown samples was added to a clear flat 96-well plate and supplemented with nitrocefin and buffer to a final volume of 100 μL. Immediately after addition of nitrocefin, absorbance at 490 nm was measured in an Envision plate reader.